普通小麦穗粒数主效QTL-qKnps-4A图位克隆及单倍型分析

Bread wheat (Triticum aestivum L.) is one of the most important food crops. Selection for high grain yield is an important objective in wheat breeding programs. Kernel number per spike (KNPS), as one of the three major yield components in wheat, is the key breeding target trait now. KNPS is quantitatively inherited and significantly influenced by the environment, which hampered the direct selection by traditional breeding methods. Mapping and cloning the genes controlling KNPS and developing the functional markers are essential for genetic improvement of wheat yield. qKnps-4A is a major stable quantitative trait locus (QTL) for KNPS, which could be repeatedly detected across multiple environments in four mapping populations. This QTL has been narrowed down to a physical distance of 0.39Mb (Chr4A:680398739–Chr4A:680789768). Positive alleles of qKnps-4A could increase no less than 3 grains per spike and in turn result in approximately 6.1% increase of yield per plant. In this project, further studies on qKnps-4A will be conducted. Secondary mapping populations for qKnps-4A have been developed, which are being used for fine mapping of qKnps-4A using a map-based cloning strategy. The candidate genes of qKnps-4A will be determined based on informations of wheat genomic sequence and bioinformatic analysis. The candidate gene is designated tentatively as TaGn-4A. The gene-expression pattern of TaGn-4A will detected by real time quantitative reverse transcription-PCR (qRT-PCR) method. Overexpression vectors, transcomplementary vectors and CRISPR/Cas9 vectors for TaGn-4A will be constructed. Transgenic approaches will be used to define the function role of TaGn-4A in wheat. Haplotype analysis of the target gene will be characterized using a natural mapping population comprised by bred varieties or advanced lines mostly from Huanghuai wheat area. The excellent haplotype of TaGn-4A will be identified. The geographical distribution of the excellent haplotype will be characterized. Functional molecular markers of TaGn-4A will be developed to facilitate genetic improvement of wheat yield. This study should be of value for further wheat molecular breeding programs designed to increase wheat yield potential.

穗粒数为小麦产量的主要构成因素，是目前育种的重要目标性状。克隆穗粒数基因，明确优异等位变异，并开发功能性分子标记，是穗粒数分子遗传改良的基础。我们前期利用4个不同分离群体检测到一个穗粒数主效QTL qKnps-4A，将其定位至4A染色体0.39Mb的物理区间），其增效等位位点增加穗粒数3.0以上，进而提高单株产量6.1%左右。在此基础上，本项目拟利用已构建的次级作图群体，采用图位克隆策略，对qKnps-4进行精细定位，结合基因组序列信息、转录组数据及比较基因组学分析，确定qKnps-4A候选基因（暂命名为TaGn-4A）。利用qRT-PCR方法检测TaGn-4A表达模式，通过转化小麦分析验证其功能。利用小麦育成品种（系）组成的自然作图群体进行单倍型分析及表型鉴定，通过候选基因关联分析明确其优异单倍型及地域分布特征，开发相关分子标记用于分子育种。

本研究对qKnps-4A进行了精细作图和候选基因分析，解析了其在小麦育种过程中的选择应用情况。基于次级作图群体基因型及表型联合鉴定分析，将qKnps-4A精细定位于KN4A: 653.72-655.91 Mb约2.19 Mb区间内，包含14个注释基因。结合参考基因组功能注释、转录组分析、荧光定量PCR检测及同源基因分析，初步推测TraesKN4A01HG38570为qKnps-4A候选基因。序列差异分析结果表明，相对于科农9204，京411中TraesKN4A01HG38570在第7外显子和第7内含子上存在两处单碱基的替换，分别造成氨基酸替换及转录本的可变剪切。时空表达分析结果显示，TraesKN4A01HG38570在NIL-KN9204小麦幼穗四个时期的表达量随着穗长增加呈先降低后升高的变化趋势，而在NIL-J411幼穗前三个时期表达量极低，在幼穗4.0 cm时期，表达量显著升高，且显著高于在NIL-KN9204中的表达量。该结果表明TraesKN4A01HG38570可能负调控穗粒数形成。基于靶基因突变体及水稻异源转化，初步证明了其对穗粒数的遗传调控功能。来自京411的优异等位基因通过增加中部小花数进而增加了中部穗粒数，最终影响了单穗总粒数，但其对小花的育性没有显著影响；此外，来自京411的qKnps-4A优异等位基因对千粒重有一定的负向效应，但在单株水平表现出正效的增产效应。育种选择效应分析显示，Hap-J411优异单倍型在国外品种及国内大部分省份品种中占比较高；在我国，随着年代的递增，Hap-J411优异单倍型类型比例呈下降趋势。但其在不同年代均表现出较高的选择效率（68.18~84.62%），表明qKnps-4A靶区段优异单倍型在现代育种过程中仍发挥着重要的作用。研究结果为qKnps-4A功能解析及其分子育种应用奠定基础，对小麦穗粒数调控机制研究和遗传改良具有重要意义。